Project Summary Most human genes are regulated after they are transcribed, at the level of splicing, mRNA decay, translation, and/or mRNA localization. The specificity of post-transcriptional regulation is driven mostly by the sequence- or structure-specific recognition of RNA by RNA-binding proteins (RBPs). We described an integrated series of efforts organized around a single but broad-reaching aim: SA1. To understand the extent, nature and evolutionary conservation of sequence context effects on RBP binding to RNA motifs. We have recently developed an assay called RNA Bind-n-Seq (RBNS) for comprehensive, quantitative analysis of an RBP?s affinity for RNA. Here, we propose to extend this approach to study the determinants of binding to natural human and mouse 3' UTR sequence by several important RBPs. Subaims are directed at understanding how RNA secondary structure impacts RBP binding to cognate motifs, understanding the effects of flanking sequence composition, assessing the conservation of RBP affinity to specific RNA regions, and developing and testing a predictive model of RBP/RNA interaction. The project is expected to yield a deeper understanding of how the sequence and structural context of an RNA motif influence its occupancy and regulatory potential, and insights into the functions of RBPs such as FMRP, hnRNP K, MBNL1 and RBFOX1 that play important roles in development and in diseases such as mental retardation, myotonic dystrophy, autism and cancer.